Novel topoisomerase II/EGFR dual inhibitors: design, synthesis and docking studies of naphtho[2′,3′:4,5]thiazolo[3,2-a]pyrimidine hybrids as potential anticancer agents with apoptosis inducing activity

Abstract Topoisomerases II are ubiquitous enzymes with significant genotoxic effects in many critical DNA processes. Additionally, epidermal growth factor receptor (EGFR) plays pivotal role in tumour growth and angiogenesis. A novel series of naphtho[2',3':4,5]thiazolo[3,2-a]pyrimidine hybrids have been designed, synthesised and evaluated for their topo IIα/EGFR inhibitory and apoptotic inducer activities. Cytotoxicity of the synthesised hybrids was evaluated against MCF-7, A549 and HCT-116 cell lines. Of the synthesised hybrids, 6i, 6a and 6c experienced superior cytotoxic activity compared to doxorubicin and erlotinib against the tested cancer cells. The molecular mechanism of these hybrids revealed their ability to successfully inhibit topo IIα and EGFR activities in micromolar concentration and may serve as topo II catalytic inhibitor. Moreover, these hybrids significantly arrested cell cycle at G2/M phase together with increased p53, caspae-7, caspase-9 levels and Bax/Bcl-2 ratio. The synthesised hybrids showed efficient binding pattern in molecular docking study and have acceptable drug likeness characters.


Introduction
Cancer is one of the most dreaded diseases and the second leading cause of death worldwide after cardiovascular diseases 1 . Despite the rapid progress in discovering novel chemotherapeutic strategies for the treatment of different types of cancer, cancer prevalence represents a rapidly aggravating problem all over the world 2 . Moreover, the lack of selectivity, efficacy, safety and multidrug resistance in currently available chemotherapeutic drugs represent hurdles against prosperity of cancer chemotherapy 3 . Consequently, there is an urgent demand to explore novel chemotherapeutic agents with high selectivity, efficacy and minimum side effects. One of the most important targets of antineoplastic drugs is DNA topoisomerases [4][5][6] .
DNA topoisomerases are essential nuclear enzymes that maintain the topological changes of DNA and play key role in catalysing DNA replication, proliferation, transcription, recombination, repair, chromosome condensation and segregation 4,7,8 . Additionally, most of available anticancer regimens based on using at least one member of topoisomerase inhibitors 6 . However, DNA topoisomerases are generally classified into topoisomerase I (topo I) and topoisomerase II (topo II) 9 . Topo I possesses its functions by cleaving only one strand of DNA, while type II cleaves both DNA strands 10 . In the same context, topo II catalysis requires two cofactors ATP and Mg þ2 in order to carry out its doublestranded DNA passage reaction 11 .
Notably, human topo II encodes two isoforms (topo Iia and topo Iib); both share 70% sequence similarity 12 . Topo Iia isoform is expressed in highly proliferating and tumour cells and implicated in cell cycle events, in contrast topo Iib isoform is predominantly expressed in non-proliferating cells such as neurons and cardiac cells 13 . Therefore, topo Iia is considered as more important target than topo Iib for the development of chemotherapeutic agents and it is already targeted by wide range of anticancer agents 14 .
Topo II inhibitor drugs can be classified according to mechanism of action into two classes 15 . The first class is topo II poison, such as doxorubicin, mitoxantrone and etoposide, which are regarded as the most important topoisomerase inhibitors especially against kidney, breast and lung cancers and they are considered as lead compounds for developing more beneficial anticancer agents [16][17][18] . This class inhibits the re-ligation step of cleaved DNA molecules resulting in DNA damage and preventing its replication and transcription 19 . The second class is topo II catalytic inhibitors, such as merbarone, novobiocin, and dexrazoxane, which act on inhibiting certain catalytic step of the topo II catalytic cycle, including DNA cleavage and prevention of ATP hydrolysis (Figure 1) 20 . Furthermore, topo II poison inhibitors suffering from tumour resistance and life-threatening toxic effects 21 . Therefore, seeking for newer topo II inhibitors are ongoing with the hope of enhanced potency and overcoming the aforementioned side effects.
Of considerable interest, protein tyrosine kinases are considered one of the most important protein families that contribute to many diseases, particularly cancer, as they are involved in almost every aspect of the cellular processes 22 . Among them, epidermal growth factor receptor (EGFR) which plays a key role in cell proliferation, survival, growth, angiogenesis, differentiation and metastasis 23 . It is over-expressed in a significant number of human tumours specifically non-small cell lung cancer (NSCLC) and breast cancer 24 . In view of this, targeting EGFR is considered an efficient goal for the development of new anti-cancer agents that inhibit tumour angiogenesis and consequently tumour growth. Several EGFR inhibitors have been reported, including the Food and Drug Administration (FDA)-approved erlotinib, lapatinib and gefitinib 25,26 (Figure 2).
Therefore, in an attempt to discover novel topo Iia/EGFR inhibitors, the present work aims to design novel series of naphtho[2 0 ,3 0 :4,5]thiazolo-[3,2-a]pyrimidine hybrids via molecular hybridisation of the two bioactive pharmacophores thiazolo [3,2a]pyrimidine and naphthoquinone into a single candidate template for the purpose of synergistic anticancer activity ( Figure 3). The synthesised hybrids have been tested as potential anticancer therapeutics against MCF-7, A549 and HCT-116 cell lines. Furthermore, hybrids eliciting promising anticancer activity were further investigated as topo Iia/EGFR inhibitors. Additionally, the cytotoxic activity of the most powerful compounds was clarified by assessing cell cycle, p53, caspases 7 and 9 levels, and Bax/Bcl2 ratios. Meanwhile, the physicochemical properties and the molecular docking study for the synthesised hybrids against the conserved ATPase domain of topo-Iia and EGFR were carried out to reveal mutual binding modes of the synthesised hybrids into DNA minor groove and specify the structural features essential for DNA recognition of these hybrids.

Synthesis of lead compounds
Chemicals and solvents used in the preparation of the target compounds are of commercial grade, and purchased from Alfa Aesar, Cambrian chemicals, Sigma-Aldrich, Acros Organics, Fluka, Merck, and El-Nasr Pharmaceutical Chemicals Companies. All chemicals were used as received.
Melting points were determined on Mel-Temp hot stage apparatus and are uncorrected. Reactions were monitored by thin-layer chromatography on Merck silica gel PF 254 plates and spots were visualised with UV light at 254 nm. IR spectra were recorded on a Bruker FT-IR spectrophotometer. 1 H NMR and 13 C NMR spectra were carried out on a Bruker apparatus operating at 400 MHz and 100 MHz, respectively using TMS as internal reference. Chemical shifts (d values) are given in parts per million (ppm) from TMS using DMSO-d 6 (2.50 ppm) as solvent and coupling constants (J) in Hz. Splitting patterns are designated as follows: s, singlet; bs, broad singlet; d, doublet; t, triplet; m, multiplet. EI-MS: Mass spectra were carried out on direct probe controller inlet part to single quadruple mass analyser in (Thermo Scientific Gcms) Model (ISQ LT) using Thermo X-Calibur Software at the Regonal Centre for Mycology and Biotechnology (RCMB) Al-Azhar University, Nasr City, Cairo. Elemental analyses were performed on Perkin-Elmer 2400 CHN Elemental analyser by the Microanalytical Centre, Faculty of Science at the University of Cairo and are within ± 0.4% of calculated values unless otherwise specified.

In vitro anti-proliferative activity
The in vitro anti-proliferative activity of the synthesised hybrids (6a-i) was evaluated using MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay, against three cancer cell lines, MCF-7, A549 and HCT-116. Cancerous cells were incubated with different concentrations of the synthesised hybrids and reference compounds (0.4 mM, 1.6 mM, 6.3 mM, 25 mM and 100 mM) for 24 h. MTT reagent, at volume equal to 10% of the culture medium volume, was added to the cultured cells and to the blank wells. The culture plates were re-incubated for 2-4 h depending on cell type and density. After the incubation period, culture plates were restored from the incubator and the obtained formazan crystals were dissolved in acidified isopropanol with continuous shaking using plate shaker (MaxQ 2000, Thermo Fisher Scientific Inc., MI, USA) at room temperature. Absorbance was measured spectrophotometrically at wavelength 570 nm using a plate reader (Stat FaxR 4200, Awareness Technology, Inc., FL, USA) [48]. Cell viability was calculated as percentage of control and the concentration that inhibits 50% of maximum cell proliferation was determined and expressed as IC 50 using Graph Pad Prism 5 software (Graph Pad software Inc., CA, USA). Three independent experiments were performed for each compound and Mean ± SE were calculated.

Topoisomerase iIa inhibitory activity
Topoisomerase iIa inhibitory activity of 6a, 6c and 6i hybrids was assessed using the human DNA topoisomerase Elisa kit (Abcam, Japan, Recombinant Human Topoisomerase II alpha protein). 6a, 6c and 6i hybrids or doxorubicin were added to the wells and incubated for 1 h at 37 C. Biotin-conjugated antibody was added to each well followed by incubation at 37 C for 1 h, then the plates were aspirated. Avidin conjugated Horseradish Peroxidase (HRP avidin) solution was added to each well, incubated again at 37 C for 1 h. TMB substrate was added to the wells and the plate was incubated at 37 C for 30 min at dark place. The absorbance was read using spectrophotometer at a wavelength of 450 nm.

DNA topo II inhibition assay
Using the procedure outlined in topoisomerase II Drug Screening Kit (Topogen, USA), the effect of 6a, 6c and 6i on topoisomerase II inhibition and the effectiveness of catenated/decatenated kDNA formation were examined. The reaction mixture includes 10 mM MgCl 2 , 50 mM Tris-HCl, 0.5 mM dithiothreitol, 30 mg/ml bovine serum albumin, 200-300 ng of kDNA and topoisomerase II. Test compounds were added to reaction mixture and etoposide used as reference standard. After the reaction samples were incubated for 30 min at 37 C, the reaction was stopped by adding 2 ml of a stop buffer containing 10% (w/v) SDS and 2 ml of 0.5 mg/ml proteinase followed by incubating the mixture for 10 min at 37 C. The reaction mixture was separated by 1% agarose gel and examined after being stained with ethidium bromide.

EGFR inhibitory assay
The kinase activity of EGFR was assessed for 6a, 6c and 6i hybrids using EGFR kinase assay kit (BPS Bioscience, San Diego, CA, USA) based on the manufacturer's instructions. Three independent experiments were performed for each compound and Mean ± SE were calculated.
Determination of Bax and Bcl-2 levels MCF-7 cells were cultured in DMEM supplied with 10% FBS and incubated in 5% CO 2 in air at 37 C. After incubation with the synthesised hybrids (6a, 6c and 6i), at their IC 50 concentrations, for 24 h, MCF-7 were lysed with extraction buffer. Cell lysate was diluted using standard diluent buffer over the range of assay. Bax and Bcl-2 content in the lysate was assessed using human Bax ELISA kit (DRG, USA) and Bcl-2 ELISA Kit (Zymed Laboratories, Invitrogen Immuno-detection, Canada), respectively, according to the manufacturer's instructions for each kit. The results were expressed as Mean ± SE of three independent experiments. Determination of p53 level p53 level was determined in MCF-7 cell line using Human p53 ELISA Kit (Sigma Aldrich, St. Louis, MO, USA). The cells were lysed using cell extraction buffer. Cell lysate was diluted with standard diluent buffer, then added to p53 monoclonal antibody precoated 96-wells plate. The plate incubated for 2 h at room temperature. Anti-p53 detection antibody was added to the plate and re-incubated at room temperature for 1 h. The plate washed four times using wash buffer followed by addition of anti-rabbit IgG-Horseradish Peroxidase (HRP) working solution. The plate incubated at room temperature for 30 min. Chromogen TMB (tetramethylbenzidine) was added to induce colour reaction. The absorbance of the plate was read at 450 nm using microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). Three independent experiments were performed for each compound and Mean ± SE were calculated.

Caspase 7 and 9 assay
The effect of 6a, 6c and 6i hybrids on caspase-7 and À9 levels in MCF-7 cell line was measured using Human Caspase-7 ELISA Kit (BioVision Inc., Milpitas, CA 95035 USA) and Human Caspase-9 ELISA Kit (EIA-4860, DRG, USA), respectively, and according to the manufacturer's instructions. The results were expressed as Mean ± SE of three independent experiments.
In vitro DNA flow Cytometry assay MCF-7 cells were seeded at density of 2 Â 10 5 into each well of a 6-well plate. The cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM), supplied with 10% foetal bovine serum, and incubated in a humidified atmosphere containing 5% CO 2 in air for 24 h at 37 C. The incubation medium was replaced with a fresh one containing the synthesised hybrids 6a and 6i at their IC 50 in DMSO (1% v/v). The cell plates were incubated for 24 h. The cells were washed twice with cold phosphate buffered saline (PBS) then fixed with 70% ice-cold ethanol. Cells washed with PBS at 37 C for 30 min and recovered by centrifugation at 2000 rpm for 5 min. Cells were stained using DNA fluorochrome propidium iodide (PI). The plates were incubated at room temperature for 20 min in a dark place. Then the cells were investigated with a FACS Calibre flow cytometer (Becton Dickinson, Heidelberg, Germany) 48 .
Annexin V-FITC/PI staining assay MCF-7 cells were incubated at a density of 4 Â 10 6 /well with synthesised compounds 6a and 6i at their IC 50 for 24 h. Cells underwent three washing cycles using ice cold PBS, then suspended in PBS. Cell apoptosis was detected by Annexin V-FITC Apoptosis Detection Kit (BioVisionResearch Products, USA). The cells were stained using PI staining solution, Annexin V-FITC and incubated for 15 min at room temperature in a dark place. Cells were investigated by flow cytometry (Ex ¼ 488 nm; Em ¼ 530 nm) using FACS calibre (Becton Dickinson, Heidelberg, Germany) 48 .
Physicochemical and pharmacokinetic properties prediction Physicochemical and pharmacokinetic properties as well as medicinal chemistry friendliness and drug-like nature of the synthesised compounds were calculated and predicted based on online pkCSM software.

Statistical analysis
Results were expressed as means ± standard error of the mean (SEM). GraphPad PrismV R was used for statistical calculations (GraphPad Software, San Diego California USA).

Molecular docking
Molecular docking study of the synthesised hybrids (6a-i), Dox and erlotinib was performed using MOE software. Ligands were built into the builder interface of the MOE program and their energies were minimised until a RMSD (root mean square deviations) gradient of 0.01 kcal/mol and RMS (Root Mean Square) distance of 0.1 Å with MMFF94Â (Merck molecular force field 94Â) force-field and the partial charges were automatically calculated. The X-ray crystallographic structure of ATP-active sites of Dox and EGFR (PDB code: 1ZXM and1XKK, respectively), were downloaded from protein data bank (www.rcsb.org). The enzymes were prepared, the hydrogens were added then the atoms connection and type were checked with automatic correction. The obtained poses were studied and the poses showed best ligand-enzyme interactions were selected and stored for energy calculations.

Result and discussion
Chemistry 6-Aryl-2-mercapto-1,6-dihydropyrimidine-5-carbonitriles 4a-i were synthesised in high yields by modified Biginelli condensation reaction 44 utilising ethyl cyanoacetate, aromatic aldehyde and thiourea in the presence of potassium carbonate as a base [45][46][47] (Scheme 1). Reaction of 4a-i with 2,3-dichloro-1,4-naphthoquinone (5) in DMF afforded the target hybrids, 2-aryl-4,6,11-trioxo-6,11-dihydro-4Hnaptho[2 0 ,3 0 :4,5]thiazolo[3,2-a]pyrimidine-3-carbonitriles 6a-i by nucleophilic attack of both NH and SH in compound 4 on both C-Cl in compound 5 under elimination of two hydrogen chloride molecules. The chemical structure of the target 6a-i hybrids was elucidated utilising the FT-IR, 1 H NMR, 13 C NMR and MS spectroscopic data as well as elemental analyses. In the IR spectra of 6a-i hybrids both significant stretching broad bands due to NH and SH at about 3250 cm À1 and 2835 cm À1 respectively, were absent. In addition, the band due to the cyano group in compound 4 did not affected by the reaction with 5. Together with, a sharp intense stretching band in the region 1701-1711 cm À1 , which advocated the presence of CO group.
In particular, the splitting pattern of the 1 H NMR spectra of 1,4naphthoquino group in thiazolopyrimidine derivative 6a, as an example, exhibited two sets of doublets at d 7.88-7.48 ppm integrated for two protons, and two sets of triplets at d 7.36-7.03 ppm for two protons. However, p-methoxyphenyl group exhibited two sets of doublets in the range 7.18-6.96 ppm integrated for four protons. The absence of any signals in the region 11.00-13.50 ppm supported the previously route explained for formation of 6a-i hybrids (Scheme 1). It has been observed that the four protons in the 1,4-naphthoquino group exhibited a multiplet and two doublets in 6c-h, however only one doublet was detected in 6b and 6i. The protons in the p-phenylene functionality exhibited, as expected, two doublets in compounds 6a,b,d-,e,h,i, however in case of 6c, the presence of two methoxy groups in positions 3 and 4 results in resonating the other three protons as follows: H-2 (s), H-5(d), H-6(d). Moreover, the two equivalent protons in symmetrically 3,4,5-trimethoxyphenyl group in 6f were detected as a singlet at 6.51 ppm, in addition to two doublets at 7.97 and 7.41 ppm, and one multiplet at 7.87 ppm.
It is worth noting that the symmetry of the un-, mono-as well as tri-substituted aryl group results in decreasing the number of signals in the 13 C NMR, so that only nineteen signals were observed for the distinctive carbons, in addition to OCH 3 and CH 3 carbon atoms. Figure 4 is a representative example for utility of 1 H and 13 C NMR spectra in structural assignment of 6i hybrid.
Additionally, the proposed structures of these compounds were confirmed by mass spectroscopic analyses, where a correct molecular ion peaks with maximum to moderate intensity were detected, which in agreement with the molecular weights of the targeted hybrids 6a-i. Also, elemental analyses confirmed the elemental compositions of these hybrids.

In vitro cytotoxic activity
In vitro cytotoxic activity of the novel synthesised hybrids (6a-i) was screened against breast carcinoma (MCF-7), lung carcinoma (A549) and colorectal carcinoma (HCT-116) human cell lines, using MTT assay. Doxorubicin (Dox) and erlotinib were used as reference drugs. The IC 50 of the synthesised hybrids was evaluated and represented in Table 1. On comparing the IC 50 values of the titled hybrids, it is clear that most of the synthesised hybrids showed strong to modest cytotoxic activity against the tested human cancer cell lines. In particular, 6i hybrid experienced the most potent cytotoxic activity among the synthesised hybrids which was evidenced by its IC 50 values of 1.79 ± 0.11 mM, 1.55 ± 0.13 mM and 2.29 ± 0.18 mM for MCF-7, A549 and HCT-116 cell lines, respectively, that represents 3.13-, 3.19-and 2.96-fold superior activity than Dox and 4.05, 3.35 and 3.77 fold superior activity than erlotinib, respectively. Similarly, 6a and 6c hybrids showed potent and efficacious cytotoxic activity greater than that achieved by both Dox and erlotinib which was cleared by their IC 50 values against the tested cancer cell lines.
Compared to Dox, 6b and 6d hybrids displayed superior cytotoxic activity against MCF-7 and A549 cell lines with IC 50 values of 4.78 ± 0.36 mM and 5.66 ± 0.29 mM for MCF-7 cell line and 4.20 ± 0.41 mM, 3.25 ± 0.31 mM for A549 cell line, respectively. Furthermore, 6b hybrid experienced potential cytotoxic activity of 1.52-and 1.23-fold the activity of erlotinib against MCF-7 and A549 cell lines, respectively. Similarly, 6d hybrid achieved 1.28and 1.60-fold the activity of erlotinib against the same cell lines. On the other hand, 6e hybrid achieved better cytotoxic activity against MCF-7 cell line with IC 50 value of 5.14 ± 0.32 mM, compared to Dox. On the other hand, 6f, 6 g and 6h hybrids showed weaker cytotoxic activity against the tested cell lines, as indicated by their IC 50 values, compared to the reference compounds. In conclusion, the preliminary results are encouraging and some of these compounds may serve as potential candidates for development of new anticancer agents.

Topoisomerase iIa inhibitory activity
Out of all the synthesised hybrids, 6a, 6c and 6i hybrids that displayed the most potent cytotoxic activity are further investigated to explore their possible mechanism of action as topoisomerase iIa inhibitor. The results were shown in Figure 5 using Dox as a reference compound. It was cleared from the results that 6a, 6c and 6i hybrids, at their IC 50 , possessed potent inhibitory activity against topoisomerase iIa. Particularly, 6i hybrid afforded the highest topoisomerase iIa inhibitory activity, where it decreases the concentration of topoisomerase iIa in MCF-7 cell line to 1.043 ± 0.10 ng/ml, representing 71.50% inhibition of topoisomerase iIa activity compared to the control MCF-7 cell line where the concentration of topoisomerase iIa was 3.663 ± 0.09 ng/ml. Moreover, 6i hybrid achieved superior topoisomerase iIa inhibitory activity compared to Dox. Dox was able to decrease the concentration of topoisomerase iIa in MCF-7 cell line to 1.248 ± 0.09 ng/ml, representing 65.92% inhibition of topoisomerase iIa activity.
Noteworthy, 6a hybrid potently reduced topoisomerase iIa concentration to 2.351 ± 0.21 ng/ml that represents 35.81% inhibition of topoisomerase iIa activity compared to control cell. Furthermore, 6c hybrid exhibited 11.30% inhibition of Figure 6. DNA topo II inhibition assay of 6i, 6a and 6c.  topoisomerase iIa activity compared to that of MCF-7 cell line. These results support that these hybrids are potent topoisomerase iIa inhibitor; and were in accordance with the in vitro cytotoxic activity of these compounds against MCF-7, A549 and HCT-116 cell lines.

DNA topo II inhibition assay
In the current assay, the catenated kDNA remained close to the well (lane 1, Figure 6), and after the addition of topo II, the catenated kDNA primarily changed to linear form (lane 2, Figure 6). When the catenated kDNA was incubated with topo II and 10 mM of 6i, 6a and 6c (lanes 3, 4 and 5, respectively, Figure 6), the production of linear DNA was not noticed and the majority of the catenated DNA stayed in the well rather than entering the gel. In conclusion, our compounds may act as catalytic inhibitors of topo II enzyme. On the other hand, a linear DNA formation was seen when catenated DNA and topo II enzyme were incubated with 10 mM etoposide (Lane 6, Figure 6) indicating that etoposide catalyses DNA breakage and inhibits DNA resealing. This ensures that etoposide serves as topo II poison.

EGFR-TK inhibitory activity
In vitro EGFR inhibitory activity of 6a, 6c and 6i hybrids were assessed and compared to the inhibitory activity of erlotinib that served as reference standard. As shown in Figure 7, 6a hybrid exhibited EGFR inhibitory activity with IC 50 values in nanomolar range of 16.03 ± 0.47 nM; while erlotinib showed relatively weaker EGFR inhibitory activity with IC 50 value of 25.68 ± 0.65 nM. Moreover, 6i hybrid showed EGFR inhibitory activity nearly equipotent to that of erlotinib. Furthermore, 6c hybrid achieved lower but remarkable anti-EGFR activity with IC 50 of 44.72 ± 0.82 nM, compared to erlotinib.
Effects of 6a, 6c and 6i hybrids on p53, Bax and Bcl-2 levels in MCF-7 cancer cell line p53 is the major tumour suppressor protein that exerts a fundamental role in preserving the integrity of human genetic information. Reduced p53 activity or mutated p53 gene are implicated in pathogenesis of majority of human cancers. In case of DNA damage or cell stress, p53 induces cell cycle arrest to expand the time required for DNA repair procedures or to induce cell apoptosis 49 . The pro-apoptotic activity of p53 is mediated by regulating the expression of several genes; which have p53 binding sites at their promoter regions. Among these genes, Bcl-2 with anti-apoptotic activity and Bax with pro-apoptotic activity 50 . p53 protein acts as up-stream regulator of Bax. Bax is located in the outer membrane of mitochondria and translocates to mitochondria at the beginning of apoptosis process, proving that it is essential for apoptosis 51 . p53 binds to Bax promotor regions and directly induces its transcription. Bax exists as homodimer or heterodimer complex with Bcl-2 50 . The BAX is deactivated upon heterodimerization with Bcl-2. Bcl-2 family regulates cell survival and prevent cell apoptosis 52 . Bcl-2 up-regulation has been implicate not only in development of many human cancers but also in metastasis and poor prognosis. Importantly, the ratio of individual members of Bcl-2 family is suggested to expose the cell to either suppressed or induced apoptosis upon exposure to certain stimuli 50 . Accordingly, modulation of relative Bax and Bcl-2 levels is fundamental in determining whether cellular apoptosis is initiated or inhibited. Accordingly, the levels of p53, Bax and Bcl-2 were measured in MCF-7 cells after treatment with our most potent cytotoxic compounds. The results were shown in Table 2, Figure 8.
Furthermore, 6a, 6c and 6i hybrids, at their IC 50 , significantly increased p53 protein level in MCF-7 cell line compared to control cells. Particularly, 6i hybrid was the most potent inducer of p53 level which was 1285 ± 29.9 pg/ml compared to 257 ± 19.1 pg/ml for control cells and 1051 ± 22.6 pg/ml for Dox treated cells. The increase in p53 level achieved by 6i hybrid represents 5 fold and 1.2 fold compared to control and Dox treated cells, respectively. Similarly, 6a and 6c hybrids achieved p53 level of 922 ± 37.5 pg/ml and 678 ± 42.3 pg/ml, respectively, that represented 3.6-fold and

2.6-fold its concentration in control cells. Additionally, Dox treatment elevated p53 level by 4-fold compared to control cells.
Notably, our data are in accordance with the results of apoptosis assays. Simultaneously, 6i hybrid was able to significantly increase the concentration of pro-apoptotic protein Bax to 581.5 ± 8.7 pg/ml compared to 83.16 ± 6.2 pg/ml for control cells, and on the top of Dox that achieved 511.2 ± 6.5 pg/ml. Moreover, 6a and 6c hybrids increased Bax concentration by 5.24 and 4.48 fold, respectively, compared to control cells.
On the other hand, the level of anti-apoptotic protein Bcl-2 was declined to 0.33 and 0.55 its level in control cells after treatment with 6c and 6i hybrids, respectively.
Effect of 6a, 6c and 6i hybrids on the levels of caspase 7 and 9 At molecular level, apoptosis is regulated via several factors, including the major tumour suppressor protein p53, Bax, Bcl-2, tumour necrosis factor-alpha (TNF-a) as well as caspases family. p53 is involved in regulation of both intrinsic and extrinsic apoptotic pathways 49 . p53 activates the extrinsic pathway by up-regulating TNF-a or Fas receptors with subsequent activation of down-stream signalling cascades that ends by recruitment of effector caspases. Assessment of these caspases serves as efficient indicator for apoptosis. Therefore, in the current study, levels of caspase 7 and 9 were measured to elucidate the effect of our compounds on MCF-7 cells on a molecular level; the results are shown in Table 3.
Notably, 6i and 6a hybrids, at their IC 50 , significantly increased caspase 7 level by 20.41-and 14.93-fold, respectively, compared to control group. Similarly, caspase 9 level was elevated by 5.38 and 4.69-fold after treatment with 6i and 6a hybrids, respectively, compared to control cells. In the same context, Dox treatment potently increased caspase 7 and 9 by 23.2-and 6.53-fold, respectively.

In vitro cell cycle analysis
In vitro DNA-flow cytometry In order to study the effect of the synthesised hybrids on cell cycle distribution and induction of apoptosis, the most cytotoxic and the most potent topo iIa/EGFR inhibitors 6a and 6i hybrids were selected to investigate their effect in MCF-7 cell line using flow cytometer analysis. The results shown in Table 4 and Figure  9(A,B) indicated that exposure of MCF-7 cell line to 6a and 6i hybrids at their IC 50 concentration (3.34 mM and 1.79 mM) induced a remarkable disruption in cell cycle profile and cell-cycle arrest.
Furthermore, the results cleared that 6a and 6i hybrids decreased cell population significantly in G0/G1 phase, where it was 36.28% and 45.94%, respectively, compared to 57.23% for control MCF-7 cell line. Additionally, the cellular population in S phase was 35.09% in control cell while it decreased to 28.94 upon treating with 6a hybrid. In G2/M phase, 6a hybrid was able to cause a significant rise in cell population to reach to 34.78% compared to 7.68% in MCF-7 cell line. Furthermore, pre-G1 phase cell population percentage extremely increased to 33.41% and 45.26% after treatment with 6a and 6i hybrids, respectively, compared to control cell (1.89%). Consequently, the results revealed that 6a and 6i hybrids significantly arrested the cell cycle in pre-G1phase and G0/G1 phase. Moreover, 6a hybrid was able to arrest the cell cycle at the G2/M phase and induce apoptosis.

Annexin V-FITC/PI staining assay
The cell cycle results cleared that 6a and 6i hybrids exhibited considerable increase in preG1 phase which is a suggestion of apoptosis. Accordingly, to ensure the apoptotic potency and to quantify the apoptosis percentage of the selected hybrids, Annexin V-FITC/propidium iodide dual staining assay was performed in MCF-7 cell line. The results in Table 5 and Figure  10(A,B) showed that the selected hybrids 6a and 6i were able to increase cell population in early apoptotic stage by 13.84 and 9.57 folds, respectively, compared to the control cell.
On the other hand, the late apoptotic cell population was significantly increased to 126.13 and 182.06 folds upon treatment with 6a and 6i hybrids, respectively, compared to the control cell. Meanwhile, 6a and 6i hybrids achieved significant increase in cell necrosis by 6.78 and 10.52 folds, respectively, compared to MCF-7 cell line. Finally, these findings evidenced the ability of the selected hybrids for inducing apoptosis at early apoptotic, late apoptotic stages as well as cell necrosis development, which in turn, confirm the ability of the synthesised hybrids to potentially inhibit topo iIa/EGFR activity via the impairment of cell division and apoptosis inducing activity.

Drug likeliness and oral bioavailability
Computational technology becomes a vital tool in drug candidate identification via contributing in optimising the therapeutic agents design and reducing the experimental drug trials required for drug selection and development. Therefore, physicochemical properties (Lipinskiʼs parameters, topological polar surface area (TPSA)), bioactivity scores and drug-likeness of the synthesised hybrids (6a-i) were predicted by using Molinspiration cheminformatics software (http://www.molinspiration.com).
According to lipinskiʼs rule of five, the molecule is considered to be orally active if it obeys the following criteria: (i) lipophilicity or the calculated octanol-water partition coefficient (LogP) 5, (ii) molecular weight (Mwt) 500, (iii) number of hydrogen bonds donors (nHBD) 5, (iv) number of hydrogen bonds acceptors (nHBA) 10. Moreover, veber's criteria involving: topological polar surface area (TPSA) < 140 Å and number of rotatable bonds (nRB)   < 10. Notably, violation of more than one of these rules indicating bioavailability problems 53 . Gratefully, the results displayed in Table  6 showed that all the synthesised hybrids possessed desirable physicochemical properties with no Lipinski property violations. Noteworthy, Log P of the studied hybrids was in the range of (2.93-4.09) indicating good lipophilicity and permeability across  the cell membrane. Additionally, these hybrids can be easily transported and absorbed through the biological membranes where the Mwt was less than 500 Da. Meanwhile, the number of hydrogen bond donors and acceptors were less than 5 and 10, respectively, which was in compliance with Lipinski's rules.
On the other hand, the results cleared that these hybrids also in accordance with Veberʼs rule, where the number of rotatable bonds was less than 10 so these hybrids had been expected to be conformationally stable as well as the TPSA of all the targeted hybrids identified in the range of (92.31-138.14 Å), suggesting good oral bioavailability. Finally, these hybrids were found to be in good agreement with the given criteria and can be said to possess promising drug-like characters with good oral bioavailability.

Bioactivity score
The bioactivity score is considered a measure of the ability of the potential drug to interact with the different receptors such as G protein-coupled receptor inspection ligand (GPCRL), ion channels, kinases, nuclear receptors, proteases and enzymes. The bioactivity scores of the studied hybrids were calculated by Molinspiration online server where if the bioactivity score is more than 0 the compound is considered to be active; if the bioactivity score is between À5.0 to 0.0 the compound is considered to be moderately active and if the score is less than À5.0 the compound is considered to be inactive [54]. On close inspection of the results tabulated in Table 7, it was cleared that the bioactivity scores of the titled hybrids (6a-i) were between À5.0 and 0.0, therefore these hybrids are considered to be moderately active towards the different measured receptors.

In silico ADMET prediction
Pharmacokinetic profile, including absorption, distribution, metabolism, excretion and toxicity, is considered an important parameter that determines the ability of the studied hybrids to act as drug leads. Therefore, pharmacokinetic features of the synthesised hybrids were predicted by using pkCSM pharmacokinetic prediction program.

Absorption studies
Many important pharmacokinetic parameters have been estimated such as Caco-2 cell permeability, human intestinal absorption (HIA), acting as P-glycoprotein substrate and P-glycoprotein I/II inhibitors. The results tabulated in Table 8 revealed that the synthesised hybrids (6a-i) exhibited good water solubility as well as moderate to high Caco-2 cell permeability indicating good in vivo absorption rate through the small intestinal wall. Moreover, human intestinal absorption (HIA%) values of all the titled hybrids were in high range from (96.5% to100%) which indicated that these hybrids could be absorbed properly from the GIT upon oral administration. Additionally, the results cleared that these hybrids act as P-glycoprotein I/II inhibitors.

Distribution studies
The distribution properties provided by pkCSM program are volume distribution (VDss), Fraction unbound (human), blood brain barrier penetration (BBB) and CNS permeability. The results in Table 9 indicated that the synthesised hybrids experienced acceptable VD, unable to penetrate the CNS and consequently poorly distributed to the brain.

Metabolism and excretion studies
The results in Table 10 cleared that all the synthesised hybrids predicted to be substrate for CYP3A4 and inhibitors for CYP1A2, CYP2C19, CYP2C9 isoforms except for 6f hybrid. Moreover, they are neither substrate for CYP2D6 or inhibitor for CYP2D6 and CYP3A4 isoforms.

Toxicity studies
The results obtained from Table 11 displayed that the estimated maximum tolerated dose for human was higher than that of Dox. Notably, the inhibition of human ether-a-go-go-related gene (hERG) cardiac potassium channel is considered a crucial factor in inhibiting cardiac arrhythmias. All the synthesised hybrids (6a-i) predicted to show inhibitory activity against hERG I and hERG II (except for 6a, 6c and 6f hybrids). Meanwhile, LD 50 value for all tested hybrids was comparable to that of Dox On the other hand,  all the studied hybrids (6a-i) exerted no hepatotoxic potential, except for 6b and 6f hybrids. These findings strongly confirm the ability of most of the titled hybrids to act as a drug.

Molecular docking study
Docking at topo iIa ATPase active site To shed light on the potential binding modes and the crucial binding features of the synthesised hybrids (6a-i) with ATPase domain of human topo iIa, molecular docking study was performed using X-ray crystallographic structure of the human topo iIa ATPase/AMP-PNP was obtained from protein databank [PDB code 1ZXM, resolution 1.87 Å (www.rcsb.org)]. Dox was used as a reference topo iIa inhibitor. The results of the binding interactions and the energy binding scores are depicted in Table 12 and Figure 11. Notably, studying T-shaped cavity of ATP binding domain found that it contains three important binding pockets: pocket I composed of various key amino acids including Asn150, Ser148, Ser149, Lys157 and Tyr34. While, pocket II contains essential amino acids including Asn91 and Asn120. Additionally, pocket III contains crucial amino acids such as Ala167, Gly166, Lys168, Tyr165, and Thr147 in addition to Mg 2þ ion 14 . Noteworthy, it was reported that the interaction with Mg 2þ ion has been considered essential for the catalytic activity at the ATPase binding domain 55 .
In close inspection of the results in Table 12 revealed the ability of nearly all of the tested hybrids to interact effectively with the key amino acids in the ATPase domain of human topoIIa compared to that of Dox. Interestingly, 6i hybrid which possessed the highest topo iIa inhibitory activity, showed efficient binding pattern with the key amino acid residues in the target protein and consequently the highest energy binding score, where it was À9.51 kcal/mol higher than that of the reference compound. Moreover, 6i hybrid was able to interact efficiently with pocket III through the formation of two hydrogen bonds between one oxygen of the free nitro group with Ala167 and Lys168. Additionally, the another oxygen of the free nitro group was able to form metal-ion interaction with Mg 2þ ion which considered one of the crucial interactions that underlying the mechanism of topoIIa inhibition where, the entry of ATP into the binding site is hindered by the interaction of the compound with the Mg 2þ ion 14 . Together with another hydrophobic interaction with Ser 149, Arg 98, Asn 91, Pro 126 and Ile 141 (Figure 11(E,F)).
Of considerable interest, 6a hybrid was engaged in a network of four H-bonds with the key amino acids in the three pockets of the target protein which have a vital role in the enhancement of topoIIa inhibitory activity. Moreover, this hybrid showed a promising binding mode as S-atom bind via H-bond with Ser 149 in pocket I, as well as, the nitrogen of the cyano group and naphthoquinone phenyl ring participated with two H-bonds with Asn 120 and Asn 91in pocket II, together with another H-bond formed between CN group and Ala 167 in pocket III (Figure 11(A,B)). On the other hand, the methoxyl oxygen and the nitrogen of the cyano group of 6c hybrid showed good fitting with pockets I and III via formation of two strong H-bonds with Ser 149 and Gly 166, respectively. Additionally, there is an important hydrophobic interaction with Asn 91 (Figure 11(C,D)).
Furthermore, the electron-withdrawing atoms in hybrids 6b, 6d and 6e caused slight decrease in the cytotoxic activity compared to the previous mentioned hybrids but they still potent enough for inhibiting the enzyme activity as their IC 50 values were nearly equipotent to that of Dox where, they able to interact with pocket I through formation of three hydrogen bonds with Ser 149, Arg 98 and Lys 157, respectively. Meanwhile, the steric factor resulting from the large size of the trimethoxy group in 6f and the dimethyl amino group in 6h hybrids negatively affects their binding pattern with the receptor site and hence their cytotoxic activity as they could not be able to bind with the crucial amino acids responsible for the activity. Finally, it can be concluded that most of the titled synthesised hybrids exhibited favourable binding interactions with topo iIa target protein in distinct manner and thus are able to inhibit its activity efficiently with pronounced cytotoxic activity.

Docking at EGFR active site
In order to predict the promising inhibitory activities of the newly synthesised hybrids against ATP active sites of EGFR kinase, the  3D crystal structure (PDB ID: 1XKK) was used in this study and erlotinib was used as a reference compound. Importantly, it was found that the key amino acid residues in EGFR kinase protein were Met 793, ASP 800, Cys797, Asp 855, Arg 841, Thr854, Asp831, Leu718, Gly721, Thr790, Phe 856 and Gly 796 [56][57][58][59] . It was cleared from the results of energy binding scores and binding interactions in Table 13 that most of the synthesised hybrids bind efficiently with the key amino acids residues in the ATP active sites of EGFR kinase.
Of considerable interest, 6a and 6i hybrids achieved better interactions with EGFR-ATP active site where the binding energy scores were higher than that of erlotinib which explained the highest experimental EGFR inhibitory activity. Additionally, the nitrogen of CN in both 6a and 6i hybrids interacts via hydrogen bonding with the key amino acid Met 793. Additionally, these hybrids participated in three hydrogen bonding interactions with Asp 855 and Lys 745 besides two p-H interactions with the key amino acid Val 726 ( Figure 12). Moreover, some hydrophobic interactions is formed with important amino acids such as Leu 718, Val 726 (for 6a hybrid) and Leu 718, Val 726, Leu 844 (for 6c hybrid). Furthermore, S and N-atoms of thiazolopyrimidine ring of 6c hybrid formed two hydrogen bonds with Asp 855 and Lys 745 in addition to p-H interaction with Val 726 and hydrophobic interactions with Arg 841, Val 726, Leu 844, Gly 721 and Thr 790 ( Figure 12).
Similarly, hydrogen bond is formed between S-atom of thiazolopyrimidine ring of 6b hybrid and Asp 855, besides two p-H interactions was formed with Val 726. However, the dimethylamino 6h hybrid formed one hydrogen bonding interaction with Lys 745 together with another p-H interactions with Leu 718. Meanwhile, the electron-withdrawing (6d and 6e hybrids), the trimethoxy 6f hybrid and the unsubstituted 6 g hybrid formed weak p-H interactions with Leu 844, Leu 718 and Val 726, respectively which was in accordance with their weak cytotoxic activity.

Structure-activity relationship
Based on the aforementioned results, it becomes obvious that the nature of the different substituents on the phenyl ring greatly influence the topoIIa/EGFR inhibitory activity and consequently the cytotoxic activity of the studied hybrids. Interestingly, 6i hybrid experienced the highest topoIIa/EGFR inhibitory activity and in turn, the highest cytotoxic activity owing to the unique ionic nature of the nitro group which was also evidenced by its higher energy binding score and its favourable binding interactions. Furthermore, the lipophilic electron donating methoxy group of 6a hybrid and dimethoxy group of 6c hybrid reflected on the efficient binding with the key amino acid residues in the receptor site resulting in good topoIIa/EGFR inhibitory activity and consequently remarkable cytotoxic activity.
Meanwhile, the hydrophilic electron-withdrawing atoms in 6b, 6d and 6e hybrids seemed to slightly decrease their cytotoxic activity compared to 6a and 6i hybrids but they still potent enough compared to IC 50 value of Dox and erlotinib which could be contributed to their ability to form hydrogen bonds with the essential amino acids of the target protein. On the contrary, the steric factor of the dimethyl amino group in 6h hybrid and the trimethoxy group of 6f hybrid hinder their proper fitting to the receptor active site and negatively affected their cytotoxic activity which was evidenced by their IC 50 values. Finally, it is possible to conclude that the electronic nature, lipophilicity, and steric factor of the substituents all have a significant impact on the biological activity of the system incorporated.

Conclusion
A novel series of naphtho[2 0 ,3 0 :4,5]thiazolo-[3,2-a]pyrimidine hybrids have been designed and synthesised as potential topo II/EGFR inhibitors. Moreover, the cytotoxic activity of the synthesised hybrids was evaluated against MCF-7, A549 and HCT-116 cell lines using Dox and erlotinib as reference compounds using MTT assay. Among the synthesised hybrids, 6i, 6a and 6c hybrids possessed superior anticancer activity of IC 50 values less than that of the reference drugs. Additionally, 6i and 6a hybrids efficiently inhibited topo iIa activity by 71.50% and 35.81%, respectively. Moreover, according to DNA topo II inhibition assay, our compounds may act as catalytic inhibitors of topo II enzyme. Furthermore, these hybrids experienced good EGFR inhibitory activity compared to erlotinib. Meanwhile, these hybrids displayed strong apoptotic activity as evident by accumulation of cells at pre-G1phase and G0/G1 phase, as well as 6a hybrid was able to arrest the cell cycle at G2/M phase and induce apoptosis. The apoptotic inducer activity of these hybrids was further confirmed by the remarkable rise in p53 level simultaneously with elevation of Bax/Bcl-2 ratio, caspase-7 and caspase-9. Importantly, the synthesised hybrids have drug likeness characters and the molecular docking study revealed that most of the synthesised hybrids possessed appropriate binding pattern with topo iIa and EGFR active sites.